Highly sensitive method for assaying chiro-inositol and compositions for the assay

ABSTRACT

The present invention relates to an assay method of chiroinositol which comprises reacting a specimen containing chiroinositol with 
     1) a dehydrogenase, which catalyses at least reversible reaction with a substrate of chiroinositol in the presence of a coenzyme selected from NAD(P)s and a coenzyme selected from thio-NAD(P)s, 
     2) A1 and 
     3) B1 
     to form cycling reaction of the formula ##STR1## wherein a product is a compound, from which 2 or 4 hydrogen atoms are deleted from chiroinositol, A1 is NAD(P)s or thio-NAD(P)s, A2 is a reduced form of A1, B1 is a reduced form of NAD(P)s in case of A1 being thio-NAD(P)s or a reduced form of thio-NAD(P)s in case of A1 being NAD(P)s and B2 is an oxidized form of B1, and determining an amount of converted A2 or B1 by the said reaction, and a composition for assay of chiroinositol. Chiroinositol can be assayed by accurate, simple, low price and high sensitive method.

FIELD OF THE INVENTION

This invention relates to high sensitive assay method of chiroinositoland a composition for assay thereof in fields of clinical biochemistryand food inspection. More particularly, this invention relates to anassay method of chiroinositol which comprises reacting a specimencontaining chiroinositol with

1) a dehydrogenase, which catalyses at least reversible reaction with asubstrate of chiroinositol in the presence of a coenzyme selected fromnicotinamide adenine dinucleotides (phosphate) [hereinafter designatesas NAD(P)s] and a coenzyme selected from thio-nicotinamide adeninedinucleotides (phosphate) [hereinafter designates as thio-NAD(P)s ]

2) A1 and

3) B1

to form cycling reaction of the formula ##STR2## wherein a product is acompound, from which 2 or 4 hydrogen atoms are deleted fromchiroinositol, A1 is NAD(P)s or thio-NAD(P)s, A2 is a reduced form ofA1, B1 is a reduced form of NAD(P)s in case of A1 being thio-NAD(P)s ora reduced form of thio-NAD(P)s in case of A1 being NAD(P)s and B2 is anoxidized form of B1, and determining an amount of converting A2 or B1 bythe said reaction. The present invention further relates to acomposition for assay of chiroinositol comprising consisting offollowing components 1)-3);

1) a dehydrogenase, which catalyses at least reversible reaction with asubstrate of chiroinositol in the presence of a coenzyme selected fromNAD(P)s and a coenzyme selected from thio-NAD(P)s,

2) at least a coenzyme selected from NAD(P)s and thio-NAD(P)s, and

3) in the above 2), at least a coenzyme selected from reduced thio-NAD(P)s in case of at least a coenzyme selected from NAD(P)s, or in theabove 2), at least a coenzyme selected from reduced NAD(P)s in case ofat least ,t coenzyme selected from thio-NAD(P)s.

PRIOR ARTS

In recent years, since an importance in insulin resistance has beenrecognized as a cause of glucose tolerance failure including diabetesmellitus, especially diabetes mellitus type II, or as a risk factor forarteriosclerosis, some of assay methods for insulin resistance havedeveloped. However, idealistic assay methods with highly accuracy,simple and low price have never been known.

Involvement of chiroinositol in insulin signal has reported and isthought to be an index for insulin resistance. [Larner J. et al., NewEng. J. Med., 323, 373-378 (1990), Published Japanese translation of PCTinternational publication for patent application (hereinafter designatesas JPCT) No. 4-504001 (WO90/10711) and JPCT No. 4-505218 (WO91/12335A)].The above JPCT No. 4-504001 and JPCT No. 4-505218 disclose that assay ofchiroinositol in body fluid such as blood or urine for diagonosis ofdiabetes mellitus, especially insulin resistance, is useful, and suggestreduction/oxidation analysis using enzyme, however no concrete methodhas proposed.

Chiroinositol measured by GC-mass spectrum analysis has been reported.[Toshimitsu Niwa, J. Chromatography, 227 (1983), 25-39, ibid, 336(1984), 345-350]. These methods require, however, pretreatment withcomplex operation and difficult to treat for many specimens as well asrequire expensive equipment, which cause high costs.

In Japanese Unexamined Patent Publication No. 8-21835, immunoassay usingspecific antibody for chiroinositol is disclosed for solving theseproblems. There may be problems, however, in the immunochemical assaywith highly sensitive in one hand and with less reproducibility,effective for treatment of specimens in a unit time and costeffectiveness as compared with biochemical assay method using enzyme inthe other hand.

For supplying accurate, simple and low cost assay method forchiroinositol, enzymatic method may be one of the most preferablemethods. However, there is no clear-cut report on an enzyme whichcatalyses a reaction with chiroinositol. Especially, no report is knownon an enzyme which catalyses a reaction with chiroinositol usingthio-NAD(P)s as coenzymes.

Myoinositol dehydrogenase produced by Aerobacter aerogenes has beenreported to have very weak activity for chiroinositol in its substratespecificity using a coenzyme NAD as compared with myoinositol (Biochim.Biophys. Acta 17, 608, 1958). Consequently, such the enzyme may not bepreferable for assay of chiroinositol, which is observed trace level invivo. Commercially available myoinositol dehydrogenase (originated fromAerobacter aerogenes, SIGMA Inc. I-0255) did not use thio-NAD(P)s as acoenzyme.

Further, there is a report that inositol dehydrogenase originated fromCryptococcus melibiosum catalyses a reaction with (+) inositol (Biochim.Biophys. Acta; 293, 295, 1973). We have cultured this strain, obtainedcrude enzyme and tried to make experiment and found that it can useNAD(P)s as a coenzyme but can not use thio-NAD(P)s. Consequently, thesaid enzyme can riot constitute cycling reaction with thio-NAD(P)s andNAD(P)s.

Problems to be solved by the invention

An object of the present invention is to provide a method for assay ofchiroinositol with accurate, simple, low cost and high sensitive methodusing an enzyme which acts on chiroinositol and a composition for assaythereof.

Means for solving problems

In order to assay trace amount of chiroinositol in vivo in clinicalbiochemical test, not only direct assay method of reduced coenzyme usingdehydrogenase but also a combination with coloring agent for assay isresulted to insufficient sensitivity. We have found that an enzymeoriginated from Bacillus sp. No. 3 has an activity for chiroinositol. Acompound generated by an action of the enzyme derived from Bacillus sp.No. 3 on chiroinositol in the presence of NAD is, for example differentfrom the case that myoinositol dehydrogenase derived from Aerobacteraerogenes acts on myoinositol in the presence of NAD to delete 2hydrogen atoms to form myoinosose 2, under sufficient progressivecondition for reaction, a compound generated from a reaction in which atfirst 2 hydrogen atoms are deleted, then 2 hydrogen atoms are furtherdeleted. A spot of the said final compound can not detected by paperchromatography [J. Biol. Chem., 241 (4); 1966, 800-806]. Since the saidfinal compound is different from myoinosose 2 and is very unstable, toconstruct a stable enzymatic cycling reaction might be impossible.

We have studied for solving the problems in this point and surprisinglyfound that the enzyme derived from Bacillus sp. No. 3 catalyzed thereaction with chiroinositol as a substrate in the presence of coenzymesthio-NAD(P)s and NAD(P)s to construct the following cycling reaction;##STR3## wherein a product is a compound, from which 2 or 4 hydrogenatoms are deleted from chiroinisitol, A1 is NAD(P)s or thio-NAD(P)s, A2is a reduced form of A1, B1 is a reduced form of NAD(P)s in case of A1being thio-NAD(P)s or a reduced form of thio-NAD(P)s in case of A1 beingNAD(P)s and B2 is an oxidized form of B1, and can be used for assay,with high sensitivity, of chiroinositol existing in vivo by assayingincreased amount of thio-NAD(P)H or decreased amount of NAD(P)H. Furtherwe have found that thio-NAD(P) can be replaced by NAD(P) as well asreplacing NAD(P)H by thio-NAD(P))H. Thereby the present invention hascompleted.

The present invention relates to an assay method of chiroinositol whichcomprises reacting a specimen containing chiroinositol with

1) a dehydrogenase, which catalyses at least reversible reaction with asubstrate of chiroinositol in the presence of a coenzyme selected fromnicotinamicle adenine dinucleotides (phosphate) [hereinafter designatesas NAD(P)s] and a coenzyme selected from thio-nicotinamide adeninedinucleotides (phosphate) [hereinafter designates as thio-NAD(P)s ],

2) A1 and

3) B1

to form cycling reaction of the formula ##STR4## wherein a product is acompound, from which 2 or 4 hydrogen atoms are deleted fromchiroinositol, A1 is NAD(P)s or thio-NAD(P)s, A2 is a reduced form ofA1, B1 is a reduced form of NAD(P)s in case of A1 being thio-NAD (P)s ora reduced form of thio-NAD(P)s in case of A1 being NAD(P)s and B2 is anoxidized form of B1, and determining an amount of converting A2 or B1 bythe said reaction. The present invention further relates to acomposition for assay of chiroinositol comprising consisting offollowing components 1)-3);

1) a dehydrogenase, which catalyses at least reversible reaction with asubstrate of chiroinositol in the presence of a coenzyme selected fromnicotinamide adenine dinucleotides (phosphate) [hereinafter designatesas NAD(P)s] and a coenzyme selected from thio-nicotinamide adeninedinucleotides (phosphate) [hereinafter designates as thio-NAD(P)s],

2) at least a coenzyme selected from NAD(P)s and thio-NAD(P)s, and

3) in the above 2), at least a coenzyme selected from reducedthio-NAD(P)s in case of at least a coenzyme selected from NAD(P)s, or inthe above 2), at least a coenzyme selected from reduced NAD(P)s in caseof at least a coenzyme selected from thio-NAD(P)s.

Preferable constituents and embodiments of the present invention areexplained in details hereinbelow.

An enzyme used in the present invention can be any of a dehydrogenase,which catalyses at least reversible reaction with a substrate ofchiroinositol in the presence of at least a coenzyme selected fromNAD(P)s and at least a coenzyme selected from thio-NAD(P)s. The enzymecan be selected by confirming the enzyme reaction using a substratechiroinositol and coenzymes consisting of a coenzyme selected fromNAD(P)s and a coenzyme selected from thio-NAD(P)s. Example of enzyme isan enzyme produced by Bacillus sp. No. 3 belonging to genus Bacillus.

The present enzyme is a specific enzyme which can act on chiroinositoland can be used coenzymes of NADs, NADPs, thio-NADs and thio-NADPs. Theenzyme is preferably used for high sensitive assay by applying enzymaticcycling using thio-NAD(P)s and NAD(P)s.

The present strain Bacillus sp. No. 3 has been deposited in the NationalInstitute of Bioscience and Human-Technology,Agency of IndustrialScience and Technology, Ministry of International Trade and Industry, inHigashi 1-1-3, Tsukuba-shi, Ibaragi-ken, Japan on Mar. 19, 1997 as FERMBP-5881. Taxonomical properties of the strain are illustratedhereinbelow.

(a) Morphological properties

Straight or slightly curved bacilli with round edges, size of0.5-0.7×1.5-3.5 μm with peritrichous movement. Elliptical to oviformsporulation with 0.8×1.0-2.0 μm in edge or subedge and expanded bysporulation. No polymorphism.

(b) Growth condition in various media

Observed findings on various media cultured at 50-52° C. for 1-2 daysare as follows.

1) Nutrient agar plat medium

Forms round with convex colonies. Smooth and round edge surface. Ocherto plae ocher in color. No formation of soluble pigment.

2) Nutrient agar slant medium

Cottony good growth. Ocher to pale ocher in color. No formation ofsoluble pigment.

3) Liquid medium (peptone water)

Good growth with uniform turbid.

4) Ltmus milk medium

Weakly acidic after 4-5 days.

(c) Physiological and biochemical properties [+: positive, +: weaklypositive, -: negative, NT: not

    ______________________________________                                        GC molar ratio % : 41.9% (HPLC)                                                 Major isoprenoid quinone : MK-7                                               Gram's stain +                                                                KOH reaction -                                                                Capsule formation -                                                           Acid-fast stain -                                                             OF test (Hugh-Leifson) NT                                                     OF test (nitrogen source : NH.sub.4 H.sub.2 PO.sub.4) F                       Aerobic growth +                                                              Anaerobic growth +                                                            Growth temperature at 70° C. -                                         60° C. +                                                               37° C. +                                                               30° C. -                                                               Halotolerant 0% +                                                             3% +                                                                          5% -                                                                          Growth pH 5.6 -                                                               6.2 -                                                                         9.0 +                                                                         Gelatin hydrolysis +                                                          Starch hydrolysis (+)                                                         Casein hydrolysis -                                                           Esculin hydrolysis +                                                          Tyrosine hydrolysis -                                                         Arginine hydrolysis -                                                         Cellulose hydrolysis -                                                        Catalase production +                                                         Oxidase production +                                                          Lecithinase production -                                                      Urease production (SSR) -                                                     Urease production (Chris) -                                                   Indole production -                                                           H.sub.2 S production (detected by lead acetate paper) -                       Acetoin production (K.sub.2 HPO.sub.4) -                                      Acetoin Production (NaCl) -                                                   MR test -                                                                     Nitrate reduction test (gas formation) -                                      (NO.sub.2 - detection) -                                                      (NO.sub.3 - detection) +                                                      Utilization on Simmons medium                                                 Citrate -                                                                     Malate -                                                                      Maleate -                                                                     Malonate -                                                                    Propionate -                                                                  Gluconate -                                                                   Succinate -                                                                   Utilization on Christensen medium                                             Citrate +                                                                     Malate -                                                                      Maleate -                                                                     Malonate -                                                                    Propionate +                                                                  Gluconate -                                                                   Succinate -                                                                   Gas formation from glucose -                                                  Acid formation from various sugars                                            Adnitol -                                                                     L (+) arabinose -                                                             Cellobiose +                                                                  Dulcitol -                                                                    Meso-erythritol -                                                             Fructose +                                                                    Fucose +                                                                      Galactose +                                                                   Glucose +                                                                     Glycerin +                                                                    Inositol +                                                                    Inulin +                                                                      Lactose +                                                                     Maltose +                                                                     Mannitol +                                                                    Mannose +                                                                     Melezitose -                                                                  Melibiose +                                                                   Raffinose -                                                                   Rhamnose +                                                                    D-ribose +                                                                    Salicin +                                                                     L-sorbose -                                                                   Sorbitol -                                                                    Starch +                                                                      Saccharose +                                                                  Trehalose +                                                                   Xylose -                                                                    ______________________________________                                    

As shown in the above, major properties of the present strain is abacilli with Gram (+) bacterium, size in 0.5-0.7×1.5-3.5 μm withperitrichous movement, with sporulation, no polymorphism, fermentativedecomposition of glucose and acid production. Catalase and oxidaseformation positive. Thermophilic facultative anaerobic. From thesecharacteristics, the present strain is referred to genus Bacillus.

Species of this strain in Bacillus is identified as follows. Accordingto Bergey's Manual of Systematic Bacteriology, Vol. 2, Bacillus species,which grow at high temperature (at 50° C.), are known 9 species of B.acidocaldarius, B. subtilis, B. badius, B. brevis, B. coagulans, B.licheniformis, B. petntothenticus, B. schegelli and B.stearothermophilus. Among them, the species which grow under anaerobiccondition are only known two species, i. e. coagulans (hereinaftersometimes designates as "C") and B. licheniformis (hereinafter sometimesdesignates as "L"). Comparison with taxonomical properties of C, L andthe present strain are shown as follows.

In the table; +: positive, (+): weakly positive, -: negative, d: differin the strain, ND: no data.

    ______________________________________                                                  C       L         the Present Strain                                ______________________________________                                        Oxidase production                                                                        -         d         +                                               Expansion with spore d - +                                                    Anaerobic growth + + +                                                        Acetoin production + + -                                                      Glucose (acid) + + +                                                          L-arabinose (acid) + + +                                                      Xylose d + -                                                                  Mannitol (acid) d + +                                                         Casein hydrolysis d + -                                                       Gelatin hydrolysis d + -                                                      Starch hydrolysis - + (+)                                                     Citrate utilization + + -                                                     Propionate utilization d + -                                                  Tylosine hydrolysis - + -                                                     LV reaction - + -                                                             Indole production - + -                                                       Halotolerance 2 % + + +                                                       5% - + -                                                                      7% - + -                                                                      10% - ND -                                                                    Growth temp. 40° C. + + +                                              50° C. + + +                                                           55° C. + + +                                                           60° C. ND ND -                                                         70° C. - - +                                                           Nitrate reduction d + -                                                       GC mole % of DNA 44.5 46.4 41.9                                                (Type) (Type)                                                                 44.3˜50.3 42.9˜49.9                                            ______________________________________                                    

As a result of the above comparison, the properties of the presentstrain is similar to those of Bacillus coagulans, however properties onacetoin production, GC molar ratio of DNA, and a reaction on litmusmilk, though not to mention in the table, are different from properties.Consequently, the present strain is referred to Bacillus sp. No. 3 inorder to differentiate from the known Bacillus species.

Further, we have found that an enzyme produced by microorganism strainssuch as Streptomyces roseogriseus 301 belonging to genus Streptomycesand Agrobacterium rhizogenes 1215 belonging to genus Agrobacterium hasan action on chiroinositol with coenzyme NAD(P)s and thio-NAD(P)s, andcan be used for high sensitive assay by applying with enzyme cyclingusing NAD(P)s and thio-NAD(P)s.

Streptomyces roseogriseus 301 has been deposited in the NationalInstitute of Biosceience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry, inHigashi 1-1-3, Tsukuba-shi, Ibaragi-ken, Japan on Feb. 26, 1998 as FERMBP-6269. Agrobacterium rhizogenes 1215 culture collection strains listNo. MAFF 301726 of the National Institute of Agrobiological Resources,in Kannondai 2-1-2, Tsukuba-shi, Ibaragi-ken, Japan, and has beendeposited in the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry, in Higashi 1-1-3, Tsukuba-shi, Ibaragi-ken, Japan onFeb. 26, 1998 as FERM BP-6270. Taxonomical properties of Streptomycesroseogriseus 301 are shown as follows.

    ______________________________________                                        Test items         Result                                                     ______________________________________                                        (a) Cell wall type     Type I                                                    LL-diaminopimeric acid +                                                      Meso-diaminopimeric acid -                                                    Diamino butyric acid -                                                        Glycine +                                                                     Aspartic acid -                                                               Ornitine -                                                                    Lysine -                                                                      Arabinose*.sup.1 -                                                            Galactose*.sup.1 -                                                           (b) Quinones MK-9 (H6), -9 (H8), -9 (H4)                                      (c) Substrate mycelia +                                                       (d) Aerial mycelia +                                                          (e) Spore chain form RF *.sup.2                                               (f) Color of colony surface gray series                                       (g) Melanin pigment formation +                                               (h) Assimilation of carbon sources                                             Arabinose +                                                                   Xylose +                                                                      Inositol +                                                                    Mannitol +                                                                    Lactose +                                                                     Rhamnose +                                                                    Sucrose +                                                                     Raffinose +                                                                ______________________________________                                         *.sup.1 Estimated by using sulfuric acid hydrolysate of whole cells.          *.sup.2 RF; rectiflexibiles (straightcurve-wavy)                         

The present strain has cell wall type 1, and from the nature of quinonesystem and a form of spore chain, it is estimated as a strain belongingto genus Streptomyces. Further this strain is estimated to belongStreptomyces roseogriseus or Streptomyces actuosus. Consequently, thepresent strain is compared with its properties to those of type strainsof Streptomyces roseogriseus IFO 13406 and Streptomyces actuosus IFO13009. Results indicate that the present strain is referred toStreptomyces roseogriseus due to similarity of spore chain length ofStreptomyces roseogriseus. The present strain is referred toStreptomyces roseogriseus 301 and deposited as FERM BP-6269.

Pure enzyme can be obtained by culturing the above Bacillus sp. No. 3,Streptomyces roseogriseus 301 or Agrobacterium rhizogenes 1215, andapplying known isolation and purification methods of proteins andenzymes. Producing microorganisms used in the present invention can bethe above mentioned Bacillus sp. No. 3, Streptomyces roseogriseus 301 orAgrobacterium rhizogenes 1215. Since the nature of bacteria can easilybe mutated in their taxonomical properties, artificial mutants, whichcan be prepared by artificial mutation means such as naturally orcommonly performed ultraviolet irradiation, radiation or treatment withmutant inducer such as N-methyl-N'-nitro-N-nitrosoguanidine or ethylmethanesulfonate, and natural mutants can be used, if such strains haveability to produce dehydrogenase having activity for reversible reactionwith a substrate of chiroinositol (hereinafter sometimes designates asdehydrogenase for chiroinositol) as well as belonging to genus Bacillus,genus Streptomyces or genus Agrobacterium, in the present invention.Further transformed bacteria, to which a gene expressing enzyme actingon chiroinositol can also be used in the present invention.

The above cultivation can be performed by applying conditions used inthe common culture of bacteria. Media used are nutrient media containingassimilable carbon sources for microorganisms, digestible nitrogensources, and if necessary inorganic sources. Examples of assimilablecarbon sources are glucose, fructose, saccharose or inositol, in asingle or in combination. Examples of digestible nitrogen sources arepeptone, meat extracts or yeast extracts, in a single or in combination.Further, salts such as phosphate, magnesium salt, calcium salt,potassium salt, sodium salt, and other heavy metal salts such as iron ormanganese can also be used, if necessary. Among others, knownassimilable carbon sources and digestible nitrogen sources can also beused.

Culture process can be performed, in general, preferably by shakeculture or aeration stirring culture under aerobic condition. Forindustrial purpose, submerged aeration culture is preferable. Culturingtemperature may be altered under conditions for growing dehydrogenase,which acts on chiroinositol, producing bacteria and producing the enzymeused in the present invention. Generally, it is preferable at 20-60° C.,specifically approximately at 50° C. for Bacillus sp. No. 3 and at 30°C. for Agrobacterium rhizogenes 1215 and Streptomyces roseogriseus 301.Culturing time can be different in the culture conditions, and thecultivation can be performed when the enzyme production reaches itsmaximum potency. In general, it is about 1-2 days. Conditions of mediumsuch as composition of medium, liquid nature of medium, culturingtemperature, stirring speed and aeration condition can be adjusted andselected in order to obtain preferable result for production. In case offoaming in the liquid culture, anti-foam agents such as silicon oil andvegetable oil can be used.

The thus obtained dehydrogenase, which has activity for chiroinositol,is an intracellular enzyme, and the cultured cells are collected byfiltration or centrifugation from the cultured mass. The cells aretreated with combination of microbial cell treatment means, for examplemechanical disruption such as by sonication, French press, glass beadsor lyophilization, or enzymatic lysis such as lysozyme, to obtain crudedehydrogenase solution having activity for chiroinositol.

Purification can be performed by combining with salting-out such asusing ammonium sulfate or sodium sulfate, chromatography such as usingmolecular sieve or ion exchange resins, electrophoresis,ultracentrifugation, desalting, heating or chemical means. Examples ofchromatography are cation or anion exchange chromatography,gel-permeation chromatography, partition chromatography, absorptionchromatography, normal phase chromatography, reverse phasechromatography, hydrophobic chromatography, hydroxyapatitechromatography or affinity chromatography. Purified enzyme can be storedin the liquid state or lyophilized state under cooling or freezing.Stabilized agents for enzyme solution or lyophilization can also beadded. Examples of stabilizing agents are sugars such as mannitol,saccharose and sorbitol, amino acid such as glutamic acid and glycine,and peptide O)r protein such as albumin.

Properties of enzyme which catalyses chiroinositol originated fromBacillus sp. No. 3 are as follows.

(1) Assay method for dehydrogenase activity for chiroinositol

(1)-1 Composition of reaction solution

    ______________________________________                                        100 mM    Tris-HCl buffer pH 8.5                                                20 mM D-chiroinositol (Wako Pure Chemicals Inc. Japan)                        2 mM NAD (Oriental Yeast Corp. Japan)                                         5 U/ml Diaphorase (Asahi Kasei Kogyo K.K., Japan)                             0.025 % NBT (Wako Pure Chemicals Inc., Japan)                                 1.5 % Triton-X 100 (Wako Pure Chemicals Inc., Japan)                        ______________________________________                                    

(1)-2 Assay of enzyme activity

The above reaction mixture 1 ml in a test tube is incubated at 37° C.for 5 minutes. Diluted enzyme solution 20 μl is added and stirred toinitiate reaction. After exactly 5 minutes, the reaction is terminatedby adding 0.1N HCl, 2 ml and stirred. A1 is obtained by measuring A550.Simultaneously, the above reaction mixture without addition ofchiroinositol is incubated by the same manner to obtain opticalabsorption A0. Enzyme activity is measured by the following equation.##EQU1## (2) Enzyme reaction

The present enzyme catalyses a reaction for generating reduced coenzyme[NAD(P)Hs and thio-NAD(P)Hs] in the presence of chiroinositol andcoenzyme [NAD(P)s and thio-NAD(P)s]. Examples of the above NAD(P)s arenicotinamide adenine dinucleotide (NAD), acetylpyridine adeninedinucleotide (acetyl NAD), nicotinamide hypoxanthine dinucleotide(deamino NAD), pyridine aldehyde adenine dinucleotide (aldehyde NAD),pyridine aldehyde hypoxanthine dinucleotide (aldehydedeamino NAD) andphosphate thereof.

Examples of the above thio-NAD(P)s are thio-nicotinamide adeninedinucleotide (thio-NAD) and thio-nicotinamide hypoxanthine dinucleotide(thio-deamino NAD) arid phosphate thereof. Initial rate of enzymereaction (originated from Bacillus sp. No. 3) are: NAD; 13 Abs/min.,acetyl NAD; 260 mAbs/min., deamino NAD; 12 Abs/min., aldehyde NAD; 140mAbs/min., NADP; 80 mAbs/min. and thio-NAD; 2.5 Abs/min.

Composition of reaction solution

    ______________________________________                                        100 mM    Gly-NaOH buffer approx. pH 10 (at optimum pH)                          10 mM D-chiroinositol (Wako Pure Chemicals Inc., Japan)                       1 mM Coenzyme (NAD(P), thio-NAD(P); Oriental Yeast                            Corp., Japan, acetyl-, aldehyde-, deamino-NAD;                                Sigma Corp., U.S.A.)                                                       ______________________________________                                    

Optimum pH are pH 11.0 for NAD, deamino NAD and thio-NAD, pH 10.5 foracetyl NAD, and pH 10.0 for aldehyde NAD and NADP.

The above reaction solution 1 ml in quartz cell is set inspectrophotometer which is controlled at 37° C. After over 5 minutesincubation, enzyme solution 140 U/ml (NAD and deamino NAD; 14 U/ml) 20μl is added and stirred. Absorption changes per minute in specificwavelength for each reduced coenzyme is measured to obtain initialreaction rate. (For NAD and deamino NAD, measured value is increasednumber by ten for adjusting the amount of enzyme.)

A product in the present cycling reaction is an amount of reduced NADgenerated by the reaction with chiroinositol and excess amount of NAD.In the reaction, 2 hydrogen atoms are deleted at the first reaction and2 hydrogen atoms are further deleted at the second reaction. These areconfirmed by increase in reduced NAD, which is determined by an amountof formazan pigment having maximum absorption at 550 nm generated as aresult of an act on of NBT (nitroblue tetrazolium) on the reduced NAD inthe presence of diaphorase.

(3) Molecular weight

135,000+10,000

Determined by gel filtration using HPLC. Column: TSK gel G3000SW(7.50×600 mm; Toso Corp., Japan), eluate; 50 mM phosphate buffer (pH7.0)+0.3 M NaCl, molecular marker; Oriental Yeast Corp., Japan.Chromatography equipment: Shimadzu Corp., Japan.

(4) Isoelectric point

pH 4.7±0.5

Focusing electrophoresis using carrier ampholites, at 4° C., constantvoltage at 700 V for 40 hours. After fractionation, activity of eachfraction is measured.

(5) Optimum pH

Approx. pH 11

Relative activity assay method hereinbelow is applied. 100 mM glycinebuffer, pH 11.0 in the reaction solution is replaced by 100 mM Trisbuffer (pH 7.5-9.0, -◯-) and 100 mM glycine buffer (pH 8.5-11.8, --).FIG. 1 shows relative activity of enzyme derived from Bacillus sp. No. 3in each pH, and at pH 11, maximum activity is observed.

Assay method for relative activity:

Composition of reaction solution

    ______________________________________                                        100 mM    glycine-NaOH buffer (pH 11.0)                                          1 mM NAD (Oriental Yeast Corp., Japan)                                        10 mM D-chiroinosiol (Wako Pure Chemicals Inc., Japan)                     ______________________________________                                    

The above reaction solution 1 ml in quartz cell is set inspectrophotometer which is (controlled at 37° C. After over 5 minutesincubation, aliquot diluted enzyme solution 20 μl is added and stirred.Absorption changes per minute at A₃₄₀ are measured to obtain initialreaction rate and to calculate relative activity.

(6) Heat stability

Enzyme solution (1 U/ml) is heat treated in 20 mM phosphate buffer (pH7.0) for 15 minutes. Remained activity is measured by the method ofassay hereinabove. FIG. 2 shows remained activities treated at varioustemperatures. Result indicates that approximately 100% activity is 55°C.

(7) Optimum temperature

Enzyme activities at 40-70° C. are assayed according to a methodhereinabove using 100 mM Tris-HCl bufer (pH 8.5). As shown in FIG. 3,maximum activity is observed at 60° C.

(8) Km value

Km values are measured at various concentrations using 10 mM

D-chiroinositol and mM NAD.

Km for D-chiroinositol 5.2±0.5 mM

Km for NAD: 0.5±0.1 mM

Km for thio-NAD is measured by replacing NAD to thio-NAD.

Km for D-chiroinositol: 12.8±0.5 mM

Km for thio-NAD: 0.9±0.1 mM

As shown in the above, the present enzyme utilizes not only NAD but alsothio-NAD as a coenzyme, and is specific enzyme having stable reactivityfor chiroinositol.

(9) Substrate specificity

Substrate specificity of the present enzyme is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Substrate     Relative activity                                               ______________________________________                                        chiroinositol 100%                                                              myoinositol 9%                                                                scylloinositol 0%                                                             epi-inositol 0%                                                               galactose 0%                                                                  fructose 0%                                                                   mannose 0%                                                                    mannitol 0%                                                                 ______________________________________                                    

Substrate specificity is assayed by using following reaction solution atthe equal concentration of substrates.

Composition of reaction solution

    ______________________________________                                        200    mM       glycine-NaOH buffer (pH 9.8)                                    0.03 mM NADH (Oriental Yeast Corp., Japan)                                    100 U/ml dehydrogenase which catalyses chiroinosiol                             (Originated from Bacillus sp. No. 3)                                        2 Mm thio-NAD (Oriental Yeast Corp., Japan)                                 ______________________________________                                    

D-chiroinositol, galactose, fructose, mannitol, mannose; Japan, WakoPure Chemicals Inc., myo-, epi-, scyllo-inositol U.S.A. SIGMA Corp.

Properties of crude enzymes which catalyses chiroinositol produced byAgrobacterium rhizogenes 1215 (hereinafter sometimes designates as A. r.1215) and Streptomyces roseogriseus 301 (hereinafter sometimesdesignates as S. r. 301) are shown as follows.

(1) Enzyme assay method

The same as in the enzyme originated from Bacillus sp. No. 3.

(2) Enzyme action

The same as in the enzyme originated from Bacillus sp. No. 3. Relativeactivities using various coenzymes are shown in Table 2 (activity usingNAD is set as 100%).

                  TABLE 2                                                         ______________________________________                                                    A.r.1215 origin                                                                        S.r.301 origin                                           ______________________________________                                        NAD           190%       100%                                                   NADP 1855% 0%                                                                 acetyl-NAD 26% 195%                                                           aldehyde-NAD 15% 20%                                                          deamino-NAD 44% 110%                                                          thio-NAD 0% 6%                                                                thio-NADP 269% 0%                                                           ______________________________________                                    

    ______________________________________                                        3) Molecular weight                                                           ______________________________________                                               A. r. 1215 origin                                                                              100,000 ± 10,000                                     S. r. 301 origin 100,000 ± 10,000                                        ______________________________________                                    

Measured by the same method as in the enzyme originated from Bacillussp. No. 3. Elution time of enzyme is determined by measuring absorptionof UV at 280 nm and activities in each fraction.

    ______________________________________                                        (4) Optimum pH                                                                ______________________________________                                                A. r. 1215 origin                                                                              approx. pH 9                                           S. r. 301 origin approx. pH 9                                               ______________________________________                                    

(5) Heat stability

Measured by the same method as in the enzyme originated from Bacillussp. No. 3.

A. r. 1215 origin: Remaining activity 100% by treatment at 40° C. for 15minutes.

S. r. 301 origin Remaining activity 100% by treatment at 50° C. for 15minutes.

(6) Km value

Km value for D-chiroinositol is shown. Measurement is performed bychanging substrate concentration in the above assay method.

A. r. 1215 origin: 30.6±1.0 mM

S. r. 301 origin: 27.6±1.0 mM

(7) Substrate specificity

Substrate specificity is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                    A.r.1215 origin                                                                        S.r.301 origin                                           ______________________________________                                        chiroinositol 100%       100%                                                   myoinositol 33% 0%                                                            scylloinositol 0% 0%                                                          epi-inositol 0% 4%                                                            fructose 0% 0%                                                                mannose 10% 0%                                                                mannitol 0% 0%                                                              ______________________________________                                    

In A. r. 1215 origin, the same assay method used in Bacillus sp. No. 3is applied by replacing coenzyme to thio-NADP. In S. r. 301 origin, thereaction solution used for enzyme assay is used by replacingD-chiroinositol for various concentrations of substrates with addingconstant concentration of enzyme solution to assay relative activities.

In order to assay extremely low concentration of chiroinositol in vivo,the enzyme cycling method shown by the following equation can generallybe used. ##STR5## wherein a product is a compound, from which 2 or 4hydrogen atoms are deleted from chiroinositol, A1 and B2 are NAD(P)s orthio-NAD(P)s, A1 and B1 are reduced form thereof, and in A1 and B1, whenA1 is thio-NAD(P)s, B1 is NAD(P)Hs, and when B1 is thio-NAD(P)H, A1 isNAD(P).

In case that dehydrogenase which catalyses chiroinositol used in anassay is required coenzyme of thio-NADs and NADs, the aforesaidthio-NADs and NADs can be selected, and in case that dehydrogenase whichcatalyses chiroinositol used in an assay is required coenzyme ofthio-NADPs and NADPs, the aforesaid thio-NADPs and NADPs can beselected.

Amount of A1 and B1 requires excess as compared with amount ofchiroinositol in the specimen, and also requires excess as compared withKm value for A1 and B1 of dehydrogenase for chiroinositol. In acomposition for assay of chiroinositol used in cycling reaction of thepresent invention, concentration of A1 and B1 is 0.02-100 mM, preferably0.05-50 mM. Amount of enzyme which catalyses chiroinositol is 1-1000U/ml, preferably 5-500 U/ml. Amount thereof can be determined preferablydepending on type and amount of specimens, consequently, the abovelevels are subject to change.

In case of enzyme cycling method in the present invention, if A1 and B1are expensive, in order to reduce amount of A1 and B1, a combination ofa dehydrogenase which constitutes a reaction of B2→B1 and not reactedwith chiroinositol or a dehydrogenase which constitutes a reaction ofA2→A1 and not reacted with chiroinositol and substrate for dehydrogenasecan be used for reducing amount of A1 and B1.

For preparation of a composition for assay of chiroinositol of thepresent invention, enzyme used for acting on chiroinositol can be adehydrogenase which uses coenzyme NAD(P)s, preferably NAD(P), andthio-NAD(P)s, preferably thio-NAD(P), and can be a dehydrogenase forassaying chiroinositol with high sensitivity by using enzyme cyclingreaction. These can be confirmed by using these coenzymes and substratebased on the knowledge of the present invention.

A composition for assay can be prepared by selecting two or morecoenzymes by considering Km value dehydrogenase for chiroinositol oneach coenzyme, and setting a condition for reaction with adjusting pHwithin optimum pH between normal reaction and reverse reaction of theenzyme cycling for effective progress of the reaction. In case ofdehydrogenase which catalyses chiroinositol from Bacillus sp. No. 3, Kmvalues for NAD and thio-NAD are small as 0.50 mM and 0.87 mM,respectiviely, cosequently thio-NAD and NAD can be selected ascoenzymes. In the cycling reaction, optimum pH of a reaction usingthio-NAD is approximately pH 10.5, and that of the revrese reactionusing NADH is approximately pH 10.0, consequently cycling can preferablybe performed by selecting coenzymes of thio-NAD and NADH at approx. pH10.

Examples of buff(er solution used are: buffer of pH 8-11 of organicamine buffer such as diethanolamine buffer, 2-ethylamino ethanol buffer,2-amino-2-methyl-1-propanol buffer and N-methyl-D-glucamine buffer, at20 mM-2 M, preferably 20 mM-1 M, most preferably 20 mM-500 mM, Good'sbuffer such as MES [2-(N-Morphilino)ethanesulfonic acid ] buffer,Bis-Tris [Bis(2-hydroxyethyl)iminotris (hydroxymethyl)methane ] buffer,ADA [N-(2-Acetamido)iminodiacetic acid ] buffer, PIPES [Piperazine-N,N'-bis(2-ethanesulfonic acid)] buffer, ACES[N-(2-Acetamido)-2-aminoethanesulfonic acid] buffer, MOPSO[3-(N-Morpholino)-2-hydroxypropanesulfonic acid ] buffer, BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] buffer, MOPS[3-(N-Morpholino)propanesulfonic acid] buffer, TES[N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer, HEPES[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid ] buffer, DIPSO {3-[N,N-Bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid} buffer,TAPSO [N-Tris(hydroxymethyl)methyl-2-hydroxy-3-aminopropanesulfonic acid] buffer, POPSO [Piperazine-N, N'-bis (2-hydroxypropanesulfonic acid ]buffer, HEPPSO [N-2-hydroxyethylpiperazine-N-hydroxypropane-3-sulfonicacid] buffer, EPPS [N-2-hydroxyethylpiperazine-N'-3-propanesulfonicacid] buffer, Tricine [Tris (hydroxymethyl) methylglycine ] buffer,Bicine [N,N-Bis(2-hydroxyethyl) glycine] buffer, TAPS [N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid] buffer, CHES[2-(Cyclohexylamino)ethanesulfonic acid] buffer, CAPSO[3-N-Cyclohexylamino-2-hydroxypropanesulfonic acid ] buffer and CAPS[3-Cyclohexylaminopropanesulfonic acid ] buffer at 20 mM-1 M, preferably20 mM-500 mM, most prederably 20 mM-300 mM, and buffers for biochemicaluse such as glycine-NaOH, potassium hydrogen phosphate-sodium dihydrogenphosphate, HCl-sodium veronal-sodium acetate, potassium hydrogenphosphate-NaOH, HCl-Tris-amino methane, HCl-amino methylpropnae diol,ammonium chloride-ammonium, borate-NaOH, HCl-sodium dimethylglycine,glycine-NaCl-HCl, disodium citrate-HCl, and sodium veronal-sodiumacetate, at 20 mM-1 M, prefer-ably 20 mM-500 mM, most preferably 20mM-300 mM. In case of using alkaline buffer, in order to make stable pHof buffer in free system, soluble hydrogen carbonate, which can releasehydrogen carbonate ion such as ,;odium hydrogen carbonate, potassiumhydrogen carbonate, magnesium hydrogen carbonate or calcium hydrogencarbonate, can be added at 10-500 mM, preferably at 50-200 mM for adjustpH. Enzyme used in these systems, single enzyme or combination of enzymecan be used.

Chiroinositol in specimens can be assayed using a composition forassaying chiroinositol of the present invention by adding specimen0.001-0.5 ml to the composition for assay of chiroinositol, reacting at37° C., and measuring an amount of generated A2 or consumed B1 based onchanges of absorption during several minutes to several tens minutesbetween 2 points after constant time from starting the reaction, forexample 1 minutes between after 3 minutes and 4 minutes, or 5 minutesbetween after 3 minutes and 8 minutes. In this case, amount ofchiroinositol in the specimens can be assayed by comparing with changesof absorption using known amount of chiroinositol.

The assay method of the present invention is constituted to introducechiroinositol per se in the specimens into enzyme cycling reaction, andis not affected by coexisting substances in the specimens, consequentlyblank asssay of the specimens can be omitted and is made possible tosimple rate assay.

Reduced coenzyme assay by measuring absorption change used in thepresent invention can be performed by other known method.

Examples of specimens containing chiroinositol are blood, urine,cerebrospinal fluid, lymph, tissue extracts and food extracts, but cannot be limited if it contains chiroinositol.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows curve of pH dependency of the enzyme produced by Bacillussp. No. 3 which catalyses chiroinositol using coenzyme NAD.

FIG. 2 shows curve of heat stability of the enzyme produced by Bacillussp. No. 3 which catalyses chiroinositol using coenzyme NAD.

FIG. 3 shows curve of optimum temperature of the enzyme produced byBacillus sp. No, 3 which catalyses chiroinositol using coenzyme NAD.

FIG. 4 shows calibration curve of chiroinositol of the presentinvention.

FIG. 5 shows calibration curve of chiroinositol of the presentinvention.

FIG. 6 shows calibration curve of chiroinositol of the presentinvention.

MODE FOR CARRYING OUT THE INVENTION

Following examples and referential examples illustrate the presentinvention in details, but are not construed as limiting the presentinvention.

REFERENTIAL EXAMPLE 1

Culture of Bacillus sp. No. 3 (National Institute of Biosceience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry, Deposition No. FERM BP-5881).

Liquid culture medium 100 ml containing yeast extract (Kyokuto SeiyakuCo., Japan) 2%, peptone (Kyokuto Seiyaku Co., Japan) 2%, potassiumdihydrogen phosphate (Wako Pure Chemicals Inc., Japan) 0.2%, calciumchloride (Wako Pure Chemicals Inc., Japan) 0.02%, magnesium sulfate(Wako Pure Chemicals Inc., Japan) 0.05% and myoinositol (Wako PureChemicals Inc., Japan) 2%, pH 7.3, was poured into 500 ml Erlenmeyer'sflask, sterized at 120° C. for 20 minutes. A loopful of Bacillus sp. No.3 (FERM BP-5881) was inoculated thereto and cultured at 50° C. for 30hours with shaking at 120 r.p.m. to obtain seed culture 85 ml (enzymeactivity 1.9 U/ml).

The liquid culture medium 20 liters, to which anti-form agent Disform442 (Nihon Yushi Co., Japan) 0.1% was added, containing the samecomposition of the above, was prepared in 30 L jar fermentor andsterilized by heating. The above seed culture 85 ml was inoculated tothe said medium and cultured at 50° C., aeration 20 L/min., innerpressure 0.4 kg/cm², agitation 150 r.p.m. for 24 hours to obtaincultures mass 18.0 L (enzyme activity 2.9 U/ml).

REFERENTIAL EXAMPLE 2

Cultured cells were collected by centrifugation of the cultured massobtained in Referential example 1, and 20 mM phosphate buffer (pH 7.5) 5L containing 0.1% lysozyme (Eisai Co., Japan) was added, then themixture was incubated at 37° C. for 1 hour. Precipitates was removed bycentrifugation to obtain supernatant solution 4.5 L (10 U/ml). Acetone1.8 L was added to the supernatant solution and stirred to collect theprecipitate by centrifugation. The thus obtained precipitated wasdissolved in 20 mM Phosphate buffer to obtain crude enzyme, 1 L (39U/ml). Solid ammonium sulfate 200 g was dissolved in the said solution,the thus formed precipitate was removed by centrifugation, and againsolid ammonium sulfate 250 g was dissolved in the thus obtainedsupernatant solution. The precipitate obtained by centrifugation of theammonium sulfate treated solution was dissolved in 20 mM phosphatebuffer (pH 7.5) to obtain enzyme solution 500 ml (58.1 U/ml). The enzymesolution was concentrated by using UF module (Asahi Kasei Kogyo K.K.,Japan, ACF-1010), desalted by using Sephadex G-25 (Pharmacia Inc.,Sweden) buffered with 20 mM phosphate buffer (pH 7.5). The solution wascharged on a column of DEAE-Sepharose CL-6B (Pharmacia Inc., Sweden) 250ml buffered with the same buffer, and developed by KCl gradient elutionof 0→0.3 M, to collect active peaks and obtain enzyme solution 400 ml(49.3 U/ml). The enzyme solution was dialyzed against 10 mM phosphatebuffer (pH 7.0) 20 L for overnight. To the enzyme solution was dissolvedbovine serum albumin (Sigma Inc., U.S.A.) 0.2 g, and lyophilized toobtain lyophilized product 1.2 g (17.0 U/mg).

REFERENTIAL EXAMPLE 3

Culture of Agrobacterium rhizogenes (National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology,Ministry of International Trade and Industry, Deposition No. FERMBP-6270) and purification of the product

Liquid culture medium 100 ml containing yeast extract (Kyokuto SeiyakuCo., Japan) 2%, peptone (Kyokuto Seiyaku Co., Japan) 2%, potassiumdihydrogen phosphate (Wako Pure Chemicals Inc., Japan) 0.2%, glucose(Kokusan Kagaku Co., Japan) 0.1% and glycerin (Wako Pure Chemicals Inc.,Japan) 2%, pH 7.0was poured into 500 ml Erlenmeyer's flask, sterilizedat 120° C. for 20 minutes. A loopful of Agrobacterium rhizogenes (FERMBP-270) was inoculated thereto and cultured at 28° C. for 30 hours withshaking at 120 r.p.m. to obtain seed culture.

The liquid culture medium 20 liters, to which anti-form agent DisformBC-51Y (Nihon Yushi Co., Japan) 0.1% was added, containing the samecomposition of the above, from which glucose and glycerin were deleted,was prepared in 30 L jar fermentor and sterilized by heating. The aboveseed culture 100 ml was inoculated to the said medium and cultured at30° C., aeration 20 L/min., inner pressure 0.4 kg/cm2, agitation 150r.p.m. for 24 hours to obtain cultured mass 20 L (enzyme activity 60mU/ml). Cultured cells were collected by centrifugation of the culturedmass, to which 20 mM phosphate buffer (pH 7.5) 2 L was added, andsonicated for 30 minutes in the ice bath. Precipitates was removed bycentrifugation t,) obtain supernatant solution 1.5 L (690 mU/ml).

The enzyme solution was charged on a column of Q-Sepharose big beads(Pharmacia Inc., Sweden) buffered with the same buffer, and stepwiselydeveloped by the same buffer containing KCl of 0, 0.1, 0.2, 0.3 and 0.4M, to collect 0.4 M fractions to obtain enzyme solution 10 L (4 mU/ml).The thus obtained enzyme solution was concentrated by using UF module(Asahi Kasei Kogyo K.K., Japan, ACP-1010) and dialyzed against 10 mMphosphate buffer (pH 7.5) 10 L for overnight to obtain crude purifiedenzyme solution 200 ml (270 mU/ml).

REFERENTIAL EXAMPLE 4

Culture of Streptomyces roseogriseus (National Institute of Bioscienceand Human-Technology, Agenct of Industrial Science and Technology,Ministry of International Trade and Industry, Deposition No. FERMBP-6269) and purification of the product

Liquid culture medium 100 ml containing beer yeast extract "Ebios"(Asahi Beer Foods Co., Japan) 2%, magnesium sulfate (Wako Pure ChemicalsInc., Japan) 0.03%, potassium dihydrogen phosphate (Wako Pure ChemicalsInc., Japan) 0.5%, glucose (Kokusan Kagaku Co., Japan) 0.1%, maltose(Nakarai Tesk Co., Japan) 1% and glycerin (Wako Pure Chemicals Inc.,Japan) 0.5%, pH 6.5 was poured into 500 ml Erlenmeyer's flask,sterilized at 120° C. for 20 minute's. A loopful of Streptomycesroseogriseus (FERM BP-6269) was inoculated thereto and cultured at 28°C. for 120 hours with shaking to obtain seed culture.

The liquid culture medium 20 liters, to which anti-form agent DisformBC-51Y (Nihon Yushi Co., Japan) 0.1% was added, containing the samecomposition of the above was prepared in 30 L jar fermentor andsterilized by heating. The above seed culture 100 ml was inoculated tothe said medium and cultured at 30° C., aeration 20 L/min., innerpressure 0.4 kg/cm², agitation 150 r.p.m. for 24 hours to obtaincultured mass 20 L (enzyme activity 60 mU/ml). Cultured cells werecollected by centrifugation of the cultured mass, to which 20 mMphosphate buffer (pH 7.5) 5 L was added, and sonicated for 30 minutes inthe ice bath. Precipitates was removed by centrifugation (7500 r.p.m.for 20 minutes) to obtain supernatant solution 3.0 L (690 mU/ml).

The enzyme solution was charged on a column of Q-Sepharose big beads(Pharmacia Inc., Sweden) 21 buffered with the same buffer, andstepwisely developed by the same buffer containing KCl of 0, 0.1, 0.2,0.3 and 0.4 M, to collect 0.2 M fractions to obtain enzyme solution 10 L(195 mU/ml). The thus obtained enzyme solution was concentrated by usingUF module (Asahi Kasei Kogyo K.K., Japan, ACP-1010) and dialyzed against10 mM phosphate buffer pH 7.5) 10 L for overnight to obtain crudepurified enzyme solution 200 ml (690 mU/ml). The obtained enzymesolution, to which NaCl (Nakarai Tesk Co., Japan) was added for 4 Mconcentration, was charged on a column of phenyl-Sepharose FF (PharmaciaInc., Sweden) 150 ml buffered with 10 mM phosphate buffer (pH 7.5)containing 4 M NaCl, developed with NaCl gradient solution of 4→0 M,pooled the active peaks to obtain the enzyme solution 360 ml (1.4 U/ml).

The obtained enzyme solution was dialyzed against 10 mM phosphate buffer(pH 7.5) 10 L for overnight, and charged on a column of DEAE-SepharoseFF (Pharmacia Inc., Sweden) 150 ml buffered with the same buffersolution, then developed with the same buffer containing 0→0.5 M of KCl,pooled the active peaks to obtain enzyme solution 100 ml (2.4 mU/ml).The obtained enzyme solution was dialyzed against 5 mM Tris-acetatebuffer (pH 6.5) 10 L for for overnight, and charged on a column ofhydroxylapatite (Asahi Pentax Co., Japan) 50 ml buffered with the samebuffer solution, then developed with the same buffer containing 0→30 mMof phosphate, pooled the active peaks to obtain enzyme solution 135 ml.The thus obtained enzyme solution was dialyzed against distilled 10 Land concentrated with membrane by which molecular weight was cut at30,000 to 25 ml (2.5 U/ml). The concentrate was freezing stored as apurified enzyme solution.

EXAMPLE 1

    ______________________________________                                        <Reaction system>                                                             ______________________________________                                        0.2  M       glycine-NaOH buffer pH 9.8                                         100 U/ml dehydrogenase which caalyses chiroinositol (Bacillus sp.                           No.3 origin)                                                    0.03 mM NADH (Oriental Yeast Corp., Japan)                                    2.0 mM thio-NAD (Oriental Yeast Corp., Japan)                               <Operation>                                                                   ______________________________________                                    

The above reagent 1 ml was set in a cuvette, added 0, 10, 35 and 50 μMof D-chiroinositol (Wako Pure Chemicals Inc., Japan), respectively, 20 aI and initiated reaction at 37° C. Absorption at 405 nm between after 1minute and 3 minutes and its difference was observed to obtain changesof absorption per minute. Results are shown in FIG. 4. As shown in FIG.4, changes of absorption for amount of chiroinositol are indicated goodlinearity.

EXAMPLE 2

    ______________________________________                                        <Reaction system>                                                             ______________________________________                                        0.2  M       glycine NaOH buffer pH 9.8                                         100 U/ml dehydrogenase which catalyses chiroinositol (Bacillus sp.                          No.3 origin)                                                    0.03 mM NADH (Oriental Yeast Corp., Japan)                                    2.0 mM thio-NADP (Oriental Yeast Corp., Japan)                              <Operation>                                                                   ______________________________________                                    

The above reagent 1 ml was set in a cuvette, added 0, 10, 20, 50, 100and 200 mM of D-chiroinositol 20 μl and initiated reaction at 37° C.Absorption al 405 nm between after 1 minute and 3 minutes and itsdifference was observed to obtain changes of absorption per minute.Results are shown in FIG. 5. As shown in FIG. 5, changes of absorptionfor amount of chiroinositol are indicated good linearity in the cyclingreaction with coenzymes thio-NADP/NADH.

EXAMPLE 3

    ______________________________________                                        <Reaction system>                                                             ______________________________________                                        0.2  M       glycine-NaOH buffer pH 9.8                                         100 U/ml dehydrogenase which catalyses chiroinositol (Bacillus sp.                          No.3 origin)                                                    0.03 mM deamino-NADH (Sigma Inc., U.S.A)                                      2.0 mM thio-NAD (Oriental Yeast Corp., Japan)                               <Operation>                                                                   ______________________________________                                    

The above reagent 1 ml was set in a cuvette, added 0, 10, 20, 35 and 50μM of D-chiroinositol 20 μl and initiated reaction at 37° C. Absorptionat 405 nm between after 1 minute and 3 minutes and its difference wasobserved to obtain changes of absorption per minute. Results are shownin FIG. 6. As shown in FIG. 6, changes of absorption for amount ofchiroinositol are indicated good linearity in the cycling reaction withcoenzymes thio-NAD/deamino-NADH.

EXAMPLE 4

    ______________________________________                                        <Reaction system>                                                             ______________________________________                                        0.2  M       glycine-NaOH buffer pH 9.8                                         100 U/ml dehydrogenase which catalyses chiroinositol (Bacillus sp.                          No.3 origin)                                                    0.03 mM NADH (Oriental Yeast Corp., Japan)                                    2.0 mM thio-NAD (Oriental Yeast Corp., Japan)                               <Operation>                                                                   ______________________________________                                    

The above reagent, 1 ml was set in a cuvette and preincubated at 37° C.Each 33 μl of urine or serum specimen and initiated reaction at 37° C.Absorption at 405 nm between after 1 minute and 3 minutes and itsdifference was observed to obtain changes of absorption per minute. Thesame operations were performed for the standard solution of 20 μMD-chiroinositol and a reagent blank, to which distilled water was addedin place of specimens. Concentration of chiroinositol in each sample wascalculated from the absorption difference of the standard solution toobtain the results shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                   difference in absorption                                                                   replaced conc. for                                      (.increment.mAbs/min) chiroinositol (μM)                                 ______________________________________                                        blank for reagent                                                                          0.3            0.0                                                 control serum 1 0.9 0.3                                                       control serum 2 1.1 0.5                                                       urine 1 46.7 34.8                                                             urine 2 14.4 10.8                                                             urine 3 131.4 98.0                                                          ______________________________________                                         In Table 4 : control serum 1 ; BML Corp. CL, Japan, and control serum 2 ;     Nissui Pharm. Co., Suitorol, Japan.                                      

EXAMPLE 5

    ______________________________________                                        <Reaction system>                                                             ______________________________________                                        280  mM      glycine-NaOH buffer pH 9.0                                         140 U/ml dehydrogenase which catalyses chiroinositol (Bacillus sp.                          No.3 origin)                                                    0.042 mM NADH (Oriental Yeast Corp., Japan)                                   2.8 mM thio-NAD (Oriental Yeast Corp., Japan)                               <Operation>                                                                   ______________________________________                                    

Aqueous solution of 1.5 M sodium carbonate (Wako Pure Chemicals Inc.,Japan) was added to the above reagent to prepare containing 300 mMsodium carbonate (pH 9.0). A reagent without containing sodium carbonatewas prepared as a control. The pH of these reagents, each 2 ml, wasmeasured immediately after preparation and after allowing to stand inthe open air at 4° C. for 110 hours.

A pH of the reagent without addition of sodium carbonate was pH 9.01immediately after preparation, whereas it was decreased to pH 8.51 (24°C.) after storage for 110 hours in the dark. The pH 9.02 thereof withaddition of sodium carbonate immediately after preparation was notchanged to pH 9.06 (24° C.) after storage for 110 hours in the dark toshow keeping stable pH value.

The activity was measured by using the above reagent according to themethod described in example 4, in which 35 μM D-chiroinositol was usedas a substrate with enzymatic cycling.

In the sodium carbonate addition group, absorption change of 38 mAb/min.was obtained, and in the control group, absorption change of 28 mAb/min.was obtained. This result indicated that the reagent containing hydrogencarbonate ion could prevent pH change as well as showing improvedability for assay of the reagent.

Effect of the invention

As shown in the above, the present invention provide rate assay of thereduced coenzyme and the blank assay for the specimen can be ommitted.Consequently, simple assay can be performed, and sensitivity of assaycan be increased by combining cycling reaction thereto. As a result,trace amount of chiroinositol in vivo can be assayed with simple andaccurate manners.

We claim:
 1. An assay method of chiroinositol which comprises reactingspecimen with1) a dehydrogenase, which catalyzes at least reversiblereaction with a substrate of chiroinositol in the presence of a coenzymeselected from nicotinamide adenine dinucleotides (phosphate),hereinafter designates as NAD(P)s and a coenzyme selected fromthio-nicotinamide adenine dinucleotides (phosphate), hereinafterdesignates as thio-NAD(P)s, 2) A1 and 3) B1 to form cycling reaction ofthe formula ##STR6## wherein a product is a compound, from which 2 or 4hydrogen atoms are deleted from chiroinositol, A1 is NAD(P)s orthio-NAD(P)s, A2 is a reduced form of A1, B1 is a reduced form ofNAD(P)s in case of A1 being thio-NAD(P)s or a reduced form ofthio-NAD(P)s in case of A1 being NAD(P)s and B2 is an oxidized form ofB1, and determining an amount of converted A2 or B1 by the saidreaction.
 2. The assay method of chiroinositol according to claim 1wherein dehydrogenase for chiroinositol is originated from Bacillus sp.No. 3 (FERM BP-5881).
 3. The assay method of chiroinositol according toclaim 1 wherein the, reaction is performed with containing solublehydrogen carbonate salt which can release hydrogen carbonate ions.
 4. Acomposition for assay of chiroinositol comprising the followingcomponents:1) a dehydrogenase, which catalyses at least reversiblereaction with a substrate of chiroinositol in the presence of a coenzymeselected from nicotinamide adenine dinucleotides (phosphate),hereinafter designates as NAD(P)s and a coenzyme selected fromthio-nicotinamide adenine dinucleotides (phosphate), hereinafterdesignates as thio-NAD(P)s, 2) at least a coenzyme selected from NAD(P)sand thio-NAD(P)s, and 3) in the above 2), at least a coenzyme selectedfrom reduced thio-NAD(P)s in case of at least a coenzyme selected fromNAD(P)s, or in the above 2), at least a coenzyme selected from reducedNAD(P)s in case of at least a coenzyme selected from thio-NAD(P)s. 5.The composition for assay of chiroinositol according to claim 4 furthercomprising soluble hydrogen carbonate salt which can release hydrogencarbonate ions.